Methods directed to crystalline biomolecules

ABSTRACT

Disclosed herein are methods of preparing a composition comprising crystalline biomolecules, for example, crystalline antibodies. In exemplary embodiments, the method comprises forming a fluidized bed of crystalline biomolecules using, for example, a counter-flow centrifuge to exchange buffer and/or to concentrate the crystalline biomolecules in a solution. Also provided are methods of detecting crystalline biomolecules and/or amorphous biomolecules in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/471,358, filed on Mar. 14, 2017, and U.S. Provisional PatentApplication No. 62/476,359, filed on Mar. 24, 2017, the contents of eachapplication are incorporated herein by reference.

BACKGROUND

Antibodies constitute powerful therapeutic agents characterized bylimited side effects due to their ability to specifically target adistinct antigen on a cell, bacteria, virus, or toxin. In 1986, thefirst therapeutic monoclonal antibody, Orthoclone OKT3, was introducedinto the market. Since then, this class of biopharmaceutical productshas significantly grown. In late 2014, 47 monoclonal antibody productshad received approval in the U.S. or Europe for the treatment of avariety of diseases, including cancer and inflammatory, cardiovascular,respiratory, and infectious diseases. Given the current approval rate ofabout four products per year, it is estimated that approximately 70monoclonal antibody products will be commercially available by 2020.See, Ecker et al., MAbs 7(1): 9-14 (2015).

Though it is reported that the projected antibody market in the UnitedStates is anticipated to increase to over $10 billion, the production ofsuch therapeutics is not without limitations. One disadvantage oftherapeutic antibodies is the cost of downstream processing to achievethe required high purity levels. While greater than 90% purity levelscan be achieved via Protein A chromatography, the cost of the adsorptionmedia is a major drawback. Another limiting factor of therapeuticantibodies is the sensitivity of antibody structure to chemical andphysical denaturation encountered during delivery and storage. Eventhough researchers have developed approaches to improve stability ofantibody formulations, some of these methods lead to a loss of proteinactivity and/or cost more due to the additional expense of proteinstabilizing carriers or formulations.

Protein crystallization is recognized in principle as an effective andscalable method of protein purification. Crystalline proteins offerhigher stability relative to their protein solution counterparts andthus have longer shelf lives. Protein purification throughcrystallization has been shown feasible by test protein products,including ovalbumin and a lipase. Insulin is the only therapeuticprotein crystallized at industrial scale. Crystallization of antibodiesis not yet routine, due to complications in their phase behavior.Precipitation, phase separation, and the formation of gel-like phasescan occur and “kinetically trap the system far from equilibrium and as aconsequence reduce the yield of crystalline protein or inhibit crystalformation completely.” See, Zang et al., PLOS One 6(9): e25282 (2011).Conventional purification techniques such as tangential flow andalternating flow filtration are not suitable for protein crystallizationpurification because of membrane fouling (i.e., the crystals clog thepores of the filter medium). Also, the high pressure required tomaintain flow through the filter can lead to excessive shear, breakage,and compaction of crystals. When the protein is an antibody, theseissues are exacerbated because the crystals of such proteins are stickyand fragile.

Thus, there is a need in the art for efficient methods of preparingcompositions comprising crystalline antibodies.

SUMMARY

Disclosed herein are methods of preparing a composition comprisingcrystalline biomolecules, for example, antigen-binding biomolecules,including, but not limited to, antibodies or immunoglobulins,antigen-binding fragments thereof, and the like. In exemplaryembodiments, the method comprises using a counter-flow gradientcentrifuge (CFGC) for exchanging the solution (e.g., buffer) in whichthe crystalline biomolecules reside and/or for concentrating thesolution (e.g., buffer) comprising the crystalline biomolecules. Thedisclosed methods are advantageous, because the steps occur in alow-shear, closed system, and under aseptic processing conditions.Multiple steps are performed in the same apparatus, thereby preventingloss of product upon transfer to different apparatuses. Due to thelow-shear environment maintained throughout the methods describedherein, the crystalline biomolecules remain separated from one another,thereby minimizing the formation of aggregates. The disclosed methodslead to attainment of a homogenous crystal slurry that can be readilypumped into downstream processing operations, such as filling.

In exemplary embodiments, the method of preparing a compositioncomprising crystalline biomolecules comprises forming a fluidized bed ofcrystalline biomolecules in a rotating chamber comprising an inlet andan outlet. Without being bound to a particular theory, it is believedthat the fluidized bed is created by rotating the chamber about asubstantially horizontal axis to create a centrifugal force(F_(centrifugal)) in said chamber, flowing a first stream of a firstsolution through the inlet in a direction opposite to the direction ofthe F_(centrifugal) and at a first flow rate (FR1) having a force(F_(FR1)) which counter balances F_(centrifugal), and collecting thefirst solution from the chamber while substantially maintaining theformation of the fluidized bed of crystallized biomolecules.

In exemplary embodiments, the method also comprises replacing the firststream of the first solution with a second stream of a second solutionwhile substantially maintaining the formation of the fluidized bed ofcrystallized biomolecules. In exemplary aspects, the method comprisesflowing a second stream of a second solution to replace the first streamof the first solution while substantially maintaining the formation ofthe fluidized bed of crystallized biomolecules. In exemplary aspects,the method comprises flowing a second stream of a second solutionthrough the inlet in a direction opposite to the direction of theF_(centrifugal) and at a flow rate (FR) equivalent to FR1 having a force(F) equal to F_(FR1), such that the force of the second stream counterbalances F_(centrifugal), and collecting the second solution from thechamber while substantially maintaining the formation of the fluidizedbed of crystallized biomolecules.

In exemplary embodiments, the method alternatively or additionallycomprises concentrating the crystallized biomolecules within a region ofthe chamber by changing FR1 to a second flow rate (FR2) having a force(F_(FR2)) which is less than F_(centrifugal) or by increasing the speedof rotation of the chamber to increase F_(centrifugal) to a level whichis greater than F_(FR1).

In exemplary aspects, the method comprises removing the crystallinebiomolecules from the chamber by, e.g., flowing a second stream into thechamber through the outlet in a direction which is parallel to thedirection of the F_(centrifugal).

Also disclosed herein are methods of detecting crystalline biomoleculesand/or amorphous biomolecules in a sample. In exemplary embodiments, themethod comprises obtaining a plurality of ¹H NMRCarr-Purcell-Meiboom-Gill (CPMG) spectra of the sample, and obtaining¹³C NMR cross-polarization (CP) spectra of the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the outlet pH profile for kSep® and a traditionalcentrifuge.

FIG. 2 is a Protein Crystallization Process Flow Diagram with the kSep®device.

FIG. 3 is a picture of a kSep® Concentrate-Wash-Harvest (CWH) graphicaluser interface.

FIG. 4 is a kSep® system piping and instrumentation diagram/drawing forharvest evaluation.

FIG. 5 is a ¹H NMR CPMG spectra of crystalline (red) vs. amorphous(blue) material.

FIG. 6 is a ¹H NMR CPMG spectra of various preparations of crystallinematerial between 100% and 0% showing the possibility of quantitation.Preparations A, B, and C contain decreasing amounts of crystals. “5%amorphous” is a preparation containing crystals with 5% amorphousmaterial and “5% crystals” is a preparation containing amorphousmaterial with 5% crystals.

FIG. 7 is a ¹H NMR CPMG spectra vs. temperature showing that theintensity of the peaks increases with temperature indicating thatmobility is increasing.

DETAILED DESCRIPTION

Disclosed herein are methods directed to crystalline biomolecules.Provided are methods for preparing a composition comprising crystallinebiomolecules. In exemplary embodiments, the method comprises forming afluidized bed of crystalline biomolecules in a rotating chambercomprising an inlet and an outlet, wherein the fluidized bed is createdby rotating the chamber about a substantially horizontal axis to createa centrifugal force (F_(centrifugal)) in said chamber, flowing a firststream of a first solution through the inlet in a direction opposite tothe direction of the F_(centrifugal) and at a first flow rate (FR1)having a force (F_(FR1)) which counter balances F_(centrifugal), andcollecting the first solution from the chamber while substantiallymaintaining the formation of the fluidized bed of crystallizedbiomolecules.

In exemplary embodiments, the method comprises a buffer exchange step, aconcentration step, or both a buffer exchange step and a concentrationstep. Further description of each step is provided below.

Buffer Exchange Step

In exemplary embodiments, the method comprises a buffer exchange stepand, in exemplary aspects, such step occurs while maintaining thefluidized bed of crystalline biomolecules. In exemplary aspects, themethod comprises replacing the first stream of the first solution with asecond stream of a second solution while substantially maintaining theformation of the fluidized bed of crystallized biomolecules. Inexemplary aspects, the method comprises flowing a second stream of asecond solution to replace the first stream of the first solution whilesubstantially maintaining the formation of the fluidized bed ofcrystallized biomolecules.

In exemplary aspects, the method comprises flowing a second stream or asecond solution through the inlet in a direction opposite to thedirection of the F_(centrifugal) and at a flow rate (FR) equivalent toFR1 having a force (F) equal to F_(FR1), such that the force of thesecond stream counter balances F_(centrifugal), and collecting thesecond solution from the chamber while substantially maintaining theformation of the fluidized bed of crystallized biomolecules. Inexemplary aspects, F_(centrifugal) is suitable to maintain thecrystalline biomolecules in the fluidized bed. In certain aspects,F_(centrifugal) is in the range of about 500 g to about 3000 g. Incertain aspects, F_(centrifugal) is in the range of about 750 g to about1250 g. In certain aspects, F_(centrifugal) is about 1000 g(±100 g)during this step.

The feed volume of the second stream of the second solution issufficient to maintain the crystalline biomolecules in the fluidizedbed. In exemplary aspects, the feed volume of the second stream of thesecond solution is about 50 to 500 ml. In exemplary aspects, the feedvolume of the second stream of the second solution is about 100 to 300ml. In exemplary aspects, the number of volumes of the first solutionper chamber is 2 or more. In exemplary aspects, FR1 is sufficient tomaintain the crystalline molecules in the fluidized bed. In exemplaryaspects, FR1 is about 50 ml/min to about 150 ml/min. In exemplaryaspects, FR1 is about 70 ml/min to about 120 ml/min.

Advantageously, the methods are not limited to a particular type ofbuffer, provided that the buffer does not negatively impact theintegrity of the crystalline biomolecules, e.g., the buffer does notnegatively impact the size, shape and/or product quality of thecrystalline biomolecules. In this regard, each of the first solution andsecond solution can be any type of buffer or solution characterized byany pH. In exemplary aspects, the pH of the solution is a physiologicalpH, e.g., 6.5 to 7.5. In exemplary aspects, the solution can have a pHthat is at least 5, at least 5.5, at least 6, at least 6.5, at least 7,at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, atleast 10, or at least 10.5 up to and including pH 11. In exemplaryaspects, one or both of the first solution and second solutionindependently comprise a buffering agent. In exemplary aspects, thebuffering agent is selected from the group consisting of: phosphatebuffers (e.g., PBS), triethanolamine, Tris, bicine, TAPS, tricine,HEPES, TES, MOPS, PIPES, cacodylate, MES, acetate, citrate, succinate,histidine or other pharmaceutically acceptable buffers.

In exemplary aspects, the first solution is a hypertonic proteincrystallization buffer. In exemplary aspects, the second solution is aformulation buffer which is safe for administration to a mammal, e.g., ahuman. In exemplary aspects, the second solution has a physiological pHand is sterile.

Concentration Step

In additional or alternative embodiments, the method comprisesconcentrating the crystallized biomolecules within a region of thechamber. In exemplary aspects, the method comprises concentrating thecrystallized biomolecules after a buffer exchange step, such as, e.g.,any of those described herein. In exemplary aspects, the concentratingstep occurs by either (i) changing FR1 to a second flow rate (FR2)having a force (F_(FR2)) which is less than F_(centrifugal) or (ii) byincreasing the speed of rotation of the chamber to increaseF_(centrifugal) to a level which is greater than F_(FR1), or (iii) both(i) and (ii). In exemplary aspects, F_(centrifugal) is 1000 g during atleast this step. In exemplary aspects, the feed volume of the secondstream of the second solution is about 100 to 500 ml. In exemplaryaspects, the feed volume of the second stream of the second solution isabout 150 ml to about 350 ml, e.g., about 150 ml, about 200 ml, about250 ml, about 300 ml, or about 350 ml. In exemplary aspects, the numberof volumes of the first solution per chamber is 1. In exemplary aspects,FR2 is about 10 ml/min to about 50 ml/min. In exemplary aspects, FR2 isless than about 60 ml/min. In exemplary aspects, FR2 is about 10 ml/minto about 50 ml/min. In exemplary aspects, FR2 is about 20 ml/min toabout 40 ml/min. In exemplary aspects, FR2 is about 20 ml/min, about 25ml/min, about 30 ml/min, about 35 ml/min, or about 40 ml/min.

In exemplary aspects, after the concentrating step, the concentration ofthe crystalline biomolecules is increased at least 2-fold, at least3-fold, or at least 4-fold.

Harvest Step

In exemplary aspects, the method comprises removing the crystallinebiomolecules from the chamber. In exemplary aspects, the methodcomprises removing the crystalline biomolecules from the chamber afterconcentrating the crystalline biomolecules within a region of thechamber. In exemplary aspects, the method comprises removing aconcentrated suspension of the crystalline biomolecules from the chamberslowly without disrupting the packed crystal suspension and/or withoutcausing cavitation within the packed crystal suspension. Cavitationwithin the packed crystal suspension is undesirable as it disrupts theconcentration profile and creates a non-uniform concentration gradientacross the concentrated crystal suspension.

In exemplary aspects, the method comprises flowing a second stream intothe chamber through the outlet in a direction which is parallel to thedirection of the F_(centrifugal) to remove the crystalline biomoleculesfrom the chamber.

In exemplary aspects, the method comprises decreasing F_(centrifugal) toa level of about 5 g to about 20 g or to a level of about 5 g to about15 g. In exemplary aspects, F_(centrifugal) is decreased to below about10 g, e.g., about 8 g.

In exemplary aspects, FR1 is controlled by a first pump and the firstpump is set to about 10 ml/min to about 100 ml/min after the bufferexchange and/or concentrating step. In exemplary aspects, FR1 iscontrolled by a first pump and the first pump is set to less than about75 ml/min. In exemplary aspects, FR1 is controlled by a first pump andthe first pump is set to about 15 ml/min to about 65 ml/min after thebuffer exchange and/or concentrating step. In exemplary aspects, thefirst pump is set to about 45 ml/min to about 65 ml/min, optionally,about 50 ml/min.

In exemplary aspects, the rotating chamber is connected to a chamberperistaltic pump. In exemplary instances, the chamber peristaltic pumpis pumping in the same direction of the first pump and F_(centrifugal).Without being bound to a particular theory, the first pump acts as a“pulling” mechanism that sucks the material out of the chamber, whereasthe chamber peristaltic pump acts as a “pushing” mechanism to ensurethere is no cavitation of the biomolecule. In exemplary aspects, thechamber peristaltic pump is set to about 50 ml/min to about 150 ml/minafter the buffer exchange and/or concentrating step. In exemplaryaspects, the chamber peristaltic pump is set to less than 100 ml/min. Insome embodiments, the chamber peristaltic pump is set to about 60 ml/minto about 90 ml/min. In exemplary instances, the chamber peristaltic pumpis set to about 75 ml/min, the first pump is set to about 50 ml/min, andF_(centrifugal) is decreased to below 10 g, optionally 8 g.

CFGC Apparatus

In exemplary aspects, the disclosed methods are carried out in acounterflow gradient centrifugation (CFGC) system. In exemplary aspects,the apparatus comprises more than one rotating chamber. In exemplaryaspects, the apparatus comprises at least 2, at least 4 or at least 6rotating chambers. In exemplary aspects, the apparatus comprises 4 or 6chambers. In exemplary embodiments, the method is carried out in anapparatus comprising more than one rotating chamber and each chamber canbe operated simultaneously with other chambers of the apparatus, or canbe operated on its own. In exemplary aspects, the steps of the disclosedmethod are carried out in more than one chamber. In exemplary aspects,the steps of the disclosed method are carried out in two, three, or fourchambers. In exemplary aspects, the steps of the disclosed method arecarried out in five or six chambers. In exemplary aspects, the steps ofthe disclosed method are carried out in more than about 6, more thanabout 10, more than about 20 chambers.

In exemplary aspects, the apparatus is a kSep® system. In exemplaryaspects, the apparatus is a kSep®400, which has four individualchambers, each of which has a 100-ml capacity. In exemplary aspects, theapparatus is a kSep®6000S, which has six individual chambers, each ofwhich has a 1000-ml capacity. The kSep®6000S has a maximum flowrate of720 L/hr.

In exemplary aspects, when the disclosed method is carried out in morethan one chamber of the apparatus, the crystalline biomolecules areremoved from the chambers one at a time. For example, the crystallinebiomolecules are removed from each chamber in a sequential manner. Thus,the removal of crystalline biomolecules from one chamber of theapparatus occurs at a time distinct from when crystalline biomoleculesare removed from another chamber of the apparatus. Without being boundto a particular theory, it is believed that removing crystallinebiomolecules from one chamber at a time reduces the chances ofcavitation of the crystalline biomolecules.

Exemplary Embodiments

In exemplary aspects, the method of preparing a composition comprisingcrystalline biomolecules comprises (a) forming a fluidized bed ofcrystalline biomolecules in a rotating chamber comprising an inlet andan outlet, wherein the fluidized bed is created by rotating the chamberabout a substantially horizontal axis to create a centrifugal force(F_(centrifugal)) in said chamber, flowing a first stream of a firstsolution through the inlet in a direction opposite to the direction ofthe F_(centrifugal) and at a first flow rate (FR1) having a force(F_(FR1)) which counter balances F_(centrifugal), and collecting thefirst solution from the chamber while substantially maintaining theformation of the fluidized bed of crystallized biomolecules, (b)performing step (i), step (ii), or step (iii), wherein step (i) isflowing a second stream of a second solution to replace the first streamof the first solution while substantially maintaining the formation ofthe fluidized bed of crystallized biomolecules, step (ii) isconcentrating the crystallized biomolecules within a region of thechamber by changing FR1 to a second flow rate (FR2) having a force(F_(FR2)) which is less than F_(centrifugal) or by increasing the speedof rotation of the chamber to increase F_(centrifugal) to a level whichis greater than F_(FR1), and step (iii) is a combination of step (i) andstep (ii), and (c) removing the crystalline biomolecules from thechamber. In exemplary aspects, the crystalline biomolecules are removedfrom the chamber by flowing a second stream into the chamber through theoutlet in a direction which is parallel to the direction of theF_(centrifugal) to remove the crystalline biomolecules from the chamber.

Additional Steps

The methods disclosed herein can comprise additional steps. For example,the methods can comprise one or more upstream steps or downstream stepsinvolved in producing, purifying, and formulating a recombinant protein.In exemplary embodiments, the method comprises steps for generating hostcells that express a recombinant protein (e.g., recombinant antibody).The host cells can be prokaryotic host cells, e.g., E. coli or Bacillussubtilis, or the host cells can be eukaryotic, e.g., yeast cells,filamentous fungi cells, protozoa cells, insect cells, or mammaliancells (e.g., CHO cells). Such host cells are described in the art. See,e.g., Frenzel, et al., Front Immunol 4: 217 (2013). For example, themethods comprise, in some instances, introducing into host cells avector comprising a nucleic acid comprising a nucleotide sequenceencoding the recombinant protein, or a polypeptide chain thereof.

In exemplary embodiments, the method comprises steps for culturing hostcells expressing a recombinant protein (e.g., recombinant antibody).Such steps are known in the art. See, e.g., Li et al., MAbs 2(5):466-477 (2010).

In exemplary embodiments, the method comprises steps for purifying therecombinant protein (e.g., recombinant antibody) from the culture. Inexemplary aspects, the method comprises one or more chromatographysteps, e.g., affinity chromatography (e.g., protein A affinitychromatography), ion exchange chromatography, and/or hydrophobicinteraction chromatography. In exemplary aspects, the method comprisessteps for producing crystalline biomolecules from a solution comprisingthe recombinant proteins. In exemplary aspects, the method comprisessteps for preparing crystalline material, including those described inInternational Patent Application Publication No. WO2016010927, entitled“CRYSTALLINE ANTIBODY FORMULATIONS” which is incorporated by referencein its entirety. The method can comprise in some aspects controlling thesolution conditions or factors which affect crystallization, e.g., therate of evaporation of solvent, organic solvents or additives, thepresence of appropriate co-solutes and buffers, pH, and temperature. Acomprehensive review of the various factors affecting thecrystallization of proteins has been published by McPherson (1985,Methods Enzymol 114: 112-120). For guidance, the teachings of McPhersonand Gilliland (1988, J Crystal Growth, 90: 51-59) includingcomprehensive lists of polypeptides that have been crystallized, as wellas the conditions under which they were crystallized, is available.Additionally, a compendium of crystals and crystallization recipes, aswell as a repository of coordinates of solved protein structures, ismaintained by the Protein Data Bank at the Brookhaven NationalLaboratory (www.rcsb.org/pdb/; Bernstein et al., 1977, J Mol Biol 112:535-542). In general, crystals are produced by combining the polypeptide(i.e., antibody) to be crystallized with an appropriate aqueous solventor aqueous solvent containing appropriate crystallization agents, suchas salts or organic solvents or additives (collectively the“crystallization reagent”). The solvent is combined with the polypeptideand can be subjected to agitation at a temperature determinedexperimentally to be appropriate for the induction of crystallizationand acceptable for the maintenance of polypeptide activity andstability. Laboratory-scale methods for crystallization include hangingdrop vapor diffusion, sitting drop vapor diffusion, microdialysis,microbatch, under oil, in gel and sandwich drop methods. The solvent canoptionally include co-crystallization additives, such as precipitants,fatty acids, reducing agents, glycerol, sulfobetaine, surfactants,polyols, divalent cations, co-factors, or chaotropes, and amino acids aswell as buffer species to control pH. “Co-crystallization additives”include compounds that facilitate crystallization of a polypeptideand/or compounds that stabilize the protein and protect againstdenaturation. Examples of co-solutes include ammonium acetate, ammoniumchloride, ammonium fluoride, ammonium formate, ammonium nitrate,ammonium phosphate, ammonium sulfate, cadmium chloride, cadmium sulfate,calcium acetate, calcium chloride, cesium chloride, cobaltous chloride,CH3(CH₂)₁₅N(CH₃)₃ Br.-(CTAB), di-ammonium citrate, di-ammonium hydrogenphosphate, di-ammonium phosphate, di-ammonium tartrate, di-potassiumphosphate, di-sodium phosphate, di-sodium tartrate, DL-malic acid,ferric chloride, L-proline, lithium acetate, lithium chloride, lithiumnitrate, lithium sulfate, magnesium acetate, magnesium chloride,magnesium formate, magnesium nitrate, magnesium sulfate, nickelchloride, potassium acetate, potassium bromide, potassium chloride,potassium citrate, potassium fluoride, potassium formate, potassiumnitrate, potassium phosphate, potassium sodium tartrate, potassiumsulfate, potassium thiocyanate, sodium acetate, sodium bromide, sodiumchloride, sodium citrate, sodium fluoride, sodium formate, sodiummalonate, sodium nitrate, sodium phosphate, sodium sulfate, sodiumthiocyanate, succinic acid, tacsimate, tri-ammonium citrate, tri-lithiumcitrate, trimethylamine N-oxide, tri-potassium citrate, tri-sodiumcitrate, zinc acetate, zinc sulfate, and other compounds that functionto supply co-solutes. “Crystallization” include compounds that maintainthe pH of a solution in a desired range to facilitate crystallization ofa polypeptide. Examples include ACES(N-(2-acetamido)-2-aminoethanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bicine(N,N-Bis(2-hydroxyethyl)glycine), BIS-TRIS(2,2-bis-(hydroxymethyl)-2,2′,2″-nitrilotriethanol), boric acid, CAPS(3-[cyclohexylamino]-1-propanesulfonic acid), citric acid, EPPS (HEPPS,4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid), Gly-Gly(NH.sub.2CH.sub.2CONHCH.sub.2COOH, glycyl-glycine), HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), imidazole, MES(2-morpholinoethanesulfonic acid), MOPS(3-(N-morpholino)-propanesulfonic acid), PIPES(piperazine-1,4-bis(2-ethanesulfonic acid)), potassium chloride, sodiumacetate, sodium bicarbonate, sodium phosphate monobasic (sodiumdihydrogen phosphate), sodium phosphate dibasic,TAPS(N-[tris-(hydroxymethyl)methyl]-3-aminopropanesulfonic acid),TAPSO(N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonicacid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid),Tricine (N-[tris(hydroxymethyl)methyl]glycine), Tris-HCl, TRIZMA(2-amino-2-(hydroxymethyl)-1,3-propanediol), and other compounds thatfunction to maintain a solution at or near a specified pH.

The selection of precipitants are one factor affecting crystallization.For example, PEG products, e.g. of molecular weight 200 to 20,000 kD,can be used. PEG3350 is a long polymer precipitant or dehydrant whichworks by volume exclusion effect. Lyotropic salts, such as ammoniumsulfate, promote precipitation processes, as do short-chain fatty acids,such as caprylic acid. Polyionic species also are useful precipitants.

Antibodies for use in formulations for subcutaneous injection, forexample, preferably are precipitated at a physiologic pH range and in acrystallization reagent that provides isotonic osmolality. The need foradditives, co-solutes, buffers, etc. and their concentrations aredetermined experimentally to facilitate crystallization.

In an industrial-scale process, controlled precipitation leading tocrystallization can best be carried out by the simple combination ofpolypeptide, precipitant, co-solutes and, optionally, buffers in a batchprocess. As another option, polypeptides can be crystallized by usingpolypeptide precipitates as the starting material (“seeding”). In thiscase, polypeptide precipitates are added to a crystallization solutionand incubated until crystals form.

Alternative laboratory crystallization methods, such as dialysis orvapor diffusion, can also be adopted. McPherson, supra and Gilliland,supra, include a comprehensive list of suitable conditions in theirreviews of the crystallization literature. Occasionally, in cases inwhich the crystallized polypeptide is to be crosslinked, incompatibilitybetween an intended crosslinking agent and the crystallization mediummight require exchanging the crystals into a more suitable solventsystem.

According to some embodiments, polypeptide crystals, crystalformulations and compositions are prepared by the following process:first, the polypeptide is crystallized. Next, excipients or ingredientsas described herein are added directly to the mother liquor.Alternatively, the crystals are suspended in a solution of excipient orother formulary ingredients, after the mother liquor is removed, for aminimum of 1 hour to a maximum of 24 hours. The excipient concentrationis typically between about 0.01 to 30% w/w, which corresponds to apolypeptide crystal concentration of 99.99 to 70% w/w, respectively. Inone embodiment, the excipient concentration is between about 0.1 to 10%,which corresponds to a crystal concentration of 99.9 to 90% w/w,respectively. The mother liquor can be removed from the crystal slurryeither by filtration, buffer exchange, or by centrifugation.

Subsequently, the crystals are washed with any isotonic injectablevehicle as long as the these vehicles do not dissolve the crystals,optionally with solutions of 50 to 100% of one or more organic solventsor additives such as, for example, ethanol, methanol, isopropanol orethyl acetate, or polyethelene glycol (PEG), either at room temperatureor at temperatures between −20° C. to 25° C. In addition, water can beused to wash the crystals. The crystals are the dried either by passinga stream of nitrogen, air, or inert gas over the crystals. Finally,micronizing of the crystals can be performed if necessary. The drying ofpolypeptide crystals is the removal of water, organic solvent oradditive, or liquid polymer by means including drying with N₂, air, orinert gases; vacuum oven drying; lyophilization; washing with a volatileorganic solvent or additive followed by evaporation of the solvent; orevaporation in a fume hood. Typically, drying is achieved when thecrystals become a free-flowing powder. Drying can be carried out bypassing a stream of gas over wet crystals. The gas can be selected fromthe group consisting of: nitrogen, argon, helium, carbon dioxide, air orcombinations thereof. The diameter of the particles achieved can be inthe range of 0.1 to 100 micrometers, or in the range of 0.2 to 10micrometers, or in the range of 10 to 50 micrometers, or in the range of0.5 to 2 micrometers. For formulations to be administered by inhalation,in one embodiment the particles formed from the polypeptide crystals arein the range of 0.5 to 1 micrometers.

According to some embodiments, when preparing protein crystals, proteincrystal formulations or compositions, enhancers, such as surfactants arenot added during crystallization. According to some other embodiments,when preparing protein crystals, protein crystal formulations orcompositions, enhancers, such as surfactants are added duringcrystallization. Excipients or ingredients are added to the motherliquor after crystallization, at a concentration of between about 1-10%w/w, alternatively at a concentration of between about 0.1-25% w/w,alternatively at a concentration of between about 0.1-50% w/w. Theseconcentrations correspond to crystal concentrations of 99-90% w/w,99.9-75% w/w and 99.9-50% w/w, respectively. The excipient or ingredientis incubated with the crystals in the mother liquor for about 0.1-3 hrs,alternatively the incubation is carried out for 0.1-12 hrs,alternatively the incubation is carried out for 0.1-24 hrs.

In some or any embodiments, the ingredient or excipient is dissolved ina solution other than the mother liquor, and the protein crystals areremoved from the mother liquor and suspended in the excipient oringredient solution. In some embodiments, the excipient or ingredientsolution (or resuspension vehicle) is a mixture of excipients oringredients or surfactants that is isotonic and injectable. In someembodiments, the excipient or ingredient solution (or resuspensionvehicle) is not a mixture of excipients or ingredients or surfactantsthat is isotonic and injectable. The ingredient or excipientconcentrations and the incubation times are the same as those describedabove.

In exemplary embodiments, the method comprises steps for formulating thepurified biomolecule (e.g., antibody). Exemplary steps are described inFormulation and Process Development Strategies for Manufacturing, eds.Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, N.J.), 2010.

In exemplary embodiments, the method comprises analyzing the sample forcrystalline vs. amorphous forms of the biomolecule. In exemplaryaspects, the method comprises quantitative analysis of the sample.

Biomolecules

As used herein, the term “biomolecule” or “biological molecule” refersto a large macromolecule that is present in living organisms or can bemade or metabolized by a living organism, or is structurally based on amolecule present in or made or metabolized by living organisms.Biomolecules include, but are not limited to being, polypeptides,proteins, polysaccharides, lipids (e.g., glycolipids, phospholipids,sterols), and polynucleotides or nucleic acids (e.g., DNA or RNA).

The methods disclosed herein are not limited to any particular type ofbiomolecule so long as the biomolecule can assume a crystalline form. Inexemplary aspects, the crystalline biomolecule is a protein comprisingone or more polypeptide chains. In exemplary aspects, the crystallineprotein is a hormone, growth factor, cytokine, a cell surface receptor,or any other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).

In exemplary instances, the biomolecule is an antibody orimmunoglobulin, or a fragment thereof, e.g., an antigen-binding antibodyfragment. As used herein, the term “antibody” refers to a protein havinga conventional immunoglobulin format, comprising heavy and light chains,and comprising variable and constant regions. For example, an antibodymay be an IgG which is a “Y-shaped” structure of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). An antibody has avariable region and a constant region. In IgG formats, the variableregion is generally about 100-110 or more amino acids, comprises threecomplementarity determining regions (CDRs), is primarily responsible forantigen recognition, and substantially varies among other antibodiesthat bind to different antigens. The constant region allows the antibodyto recruit cells and molecules of the immune system. The variable regionis made of the N-terminal regions of each light chain and heavy chain,while the constant region is made of the C-terminal portions of each ofthe heavy and light chains. (Janeway et al., “Structure of the AntibodyMolecule and the Immunoglobulin Genes”, Immunobiology: The Immune Systemin Health and Disease, 4^(th) ed. Elsevier Science Ltd./GarlandPublishing, (1999)).

The general structure and properties of CDRs of antibodies have beendescribed in the art. Briefly, in an antibody scaffold, the CDRs areembedded within a framework in the heavy and light chain variable regionwhere they constitute the regions largely responsible for antigenbinding and recognition. A variable region typically comprises at leastthree heavy or light chain CDRs (Kabat et al., 1991, Sequences ofProteins of Immunological Interest, Public Health Service N.I.H.,Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342: 877-883), within aframework region (designated framework regions 1-4, FR1, FR2, FR3, andFR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

Antibodies can comprise any constant region known in the art. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgGhas several subclasses, including, but not limited to IgG1, IgG2, IgG3,and IgG4. IgM has subclasses, including, but not limited to, IgM1 andIgM2. Embodiments of the invention include all such classes or isotypesof antibodies. The light chain constant region can be, for example, akappa- or lambda-type light chain constant region, e.g., a human kappa-or lambda-type light chain constant region. The heavy chain constantregion can be, for example, an alpha-, delta-, epsilon-, gamma-, ormu-type heavy chain constant regions, e.g., a human alpha-, delta-,epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly,in exemplary embodiments, the antibody is an antibody of isotype IgA,IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

The antibody may be a monoclonal antibody or a polyclonal antibody. Insome embodiments, the antibody comprises a sequence that issubstantially similar to a naturally-occurring antibody produced by amammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, andthe like. In this regard, the antibody may be considered as a mammalianantibody, e.g., a mouse antibody, rabbit antibody, goat antibody, horseantibody, chicken antibody, hamster antibody, human antibody, and thelike. In certain aspects, the biomolecule is an antibody, such as ahuman antibody. In certain aspects, the biomolecule is a chimericantibody or a humanized antibody. The term “chimeric antibody” refers toan antibody containing domains from two or more different antibodies. Achimeric antibody can, for example, contain the constant domains fromone species and the variable domains from a second, or more generally,can contain stretches of amino acid sequence from at least two species.A chimeric antibody also can contain domains of two or more differentantibodies within the same species. The term “humanized” when used inrelation to antibodies refers to antibodies having at least CDR regionsfrom a non-human source which are engineered to have a structure andimmunological function more similar to true human antibodies than theoriginal source antibodies. For example, humanizing can involve graftinga CDR from a non-human antibody, such as a mouse antibody, into a humanantibody. Humanizing also can involve select amino acid substitutions tomake a non-human sequence more similar to a human sequence.

An antibody can be cleaved into fragments by enzymes, such as, e.g.,papain and pepsin. Papain cleaves an antibody to produce two Fabfragments and a single Fc fragment. Pepsin cleaves an antibody toproduce a F(ab′)₂ fragment and a pFc′ fragment. In exemplary aspects,the biomolecule can be an antibody fragment, e.g., a Fab, Fc, F(ab′)₂,or a pFc′, that retains at least one glycosylation site.

The architecture of antibodies has been exploited to create a growingrange of alternative antibody formats that spans a molecular-weightrange of at least about 12-150 kDa and has a valency (n) range frommonomeric (n=1), to dimeric (n=2) and to trimeric (n=3) to tetrameric(n=4) and potentially higher; such alternative antibody formats arereferred to herein as “antibody protein products”.

Antibody protein products include those based on antibody fragments,e.g., scFvs, Fabs and VHH/VH (discussed below), which retain fullantigen-binding capacity. The smallest antigen-binding fragment thatretains its complete antigen binding site is the Fv fragment, whichconsists entirely of variable (V) regions. A soluble, flexible aminoacid peptide linker is used to connect the V regions to a scFv (singlechain fragment variable) fragment for stabilization of the molecule, orthe constant (C) domains are added to the V regions to generate a Fabfragment [fragment, antigen-binding]. Both scFv and Fab fragments can beeasily produced in host cells, e.g., prokaryotic host cells. Otherantibody protein products include disulfide-bond stabilized scFv(ds-scFv), single chain Fab (scFab), as well as di- and multimericantibody formats like dia-, tria- and tetra-bodies, or minibodies(miniAbs) that comprise different formats consisting of scFvs linked tooligomerization domains. The smallest fragments are VHH/VH of camelidheavy chain Abs as well as single domain Abs (sdAb). The building blockthat is most frequently used to create novel antibody formats is thesingle-chain variable (V)-domain antibody fragment (scFv), whichcomprises V domains from the heavy and light chain (VH and VL domain)linked by a peptide linker of ˜15 amino acid residues. A peptibody orpeptide-Fc fusion is yet another antibody protein product. The structureof a peptibody consists of a biologically active peptide grafted onto anFc domain. Peptibodies are well-described in the art. See, e.g.,Shimamoto et al., mAbs 4(5): 586-591 (2012).

Other antibody protein products include a single chain antibody (SCA); adiabody; a triabody; a tetrabody; bispecific or trispecific antibodies,and the like. Bispecific antibodies can be divided into five majorclasses: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteinsand BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology67(2) Part A: 97-106 (2015).

In exemplary aspects, the biomolecule comprises any one of theseantibody protein products. In exemplary aspects, the biomolecule is anyone of an scFv, Fab VHH/VH, Fv fragment, ds-scFv, scFab, dimericantibody, multimeric antibody (e.g., a diabody, triabody, tetrabody),miniAb, peptibody VHH/VH of camelid heavy chain antibody, sdAb, diabody;a triabody; a tetrabody; a bispecific or trispecific antibody, BsIgG,appended IgG, BsAb fragment, bispecific fusion protein, and BsAbconjugate.

The biomolecule may be an antibody protein product in monomeric form, orpolymeric, oligomeric, or multimeric form. In certain embodiments inwhich the antibody comprises two or more distinct antigen bindingregions fragments, the antibody is considered bispecific, trispecific,or multi-specific, or bivalent, trivalent, or multivalent, depending onthe number of distinct epitopes that are recognized and bound by theantibody.

Methods of Detecting Crystalline and Amorphous Biomolecules

To ensure a robust and safe formulation, the extent of crystallinity ordisorder is optionally monitored during various processing steps (e.g.,bulk material scale-up, formulation development, manufacturing), andthroughout the shelf-life of a pharmaceutical product. Amorphous formsof pharmaceutical drug substances along with their downstreamconsequences in drug products and biological systems is well documented.See, e.g., Shah et al., J Pharm Sci 95(8): 1641 (2006). An amorphoussolid is usually defined in reference to a crystalline solid andtypically lacks long-range translational orientation symmetry that ischaracteristic of crystalline structures. The amorphous phase can occurthroughout the particle or in parts of the particle, such as theparticle's surface. Detection of amorphous forms of a solid can bechallenging, as the disorder can be too small to easily detect.Sufficiently large amounts of disorder can cause changes in productperformance, affecting, e.g., post-compression hardness, enhanceddissolution rate, reduced chemical stability, and moisture-inducedrecrystallization during storage. See, Shah et al., 2006, supra. Variousquantification techniques exist to detect amorphous forms of a solid(relative to a crystalline solid), and include, e.g., powder X-raydiffraction (PXRD), differential scanning calorimetry (DSC), isothermalmicrocalorimetry (IMC), solution calorimetry (SC), infrared spectroscopy(IRR, Fourier-Transform (FT) Raman Spectroscopy, and solid-state NMR(ssNMR). However, each has one or more disadvantages.

In addition to the methods of preparing a composition comprisingcrystalline biomolecules, provided herein are efficient and facilemethods for detecting crystalline biomolecules and/or amorphousbiomolecules in a sample.

In exemplary embodiments, the method of detecting crystallinebiomolecules and/or amorphous biomolecules in a sample comprisesperforming high resolution ssNMR (solid state Nuclear MagneticResonance) analysis on the sample. In exemplary embodiments, the methodcomprises obtaining high resolution ¹³C ssNMR spectra using protondecoupling and magic angle spinning (MAS), wherein sensitivityenhancement is achieved by cross-polarization (CP). In exemplaryaspects, the method of detecting crystalline biomolecules and/oramorphous biomolecules in a sample comprises obtaining a plurality of ¹HCarr-Purcell-Meiboom-Gill (CPMG) spectra of the sample and obtaining a¹³C cross-polarization (CP) spectra of the sample. In exemplary aspects,the method comprises operating a ¹H resonance frequency of about 250 toabout 1000 MHz (optionally, about 500 MHz, about 700 MHz, about 800 MHz,about 900 MHz). In exemplary aspects, the method comprises maintainingthe temperature at about 250 to 350 K, e.g., about 250 K, about 275 K,about 300 K, about 325 K, or about 350 K. In exemplary aspects, themethod comprises operating a magic angle spinning (MAS) probe. Inexemplary instances, the method comprises operating a MAS probecomprising at least two radio frequency (rf) channels. In exemplaryaspects, the MAS probe is tuned to ¹H and ¹³C. In certain aspects, theMAS probe is operated with a spinning frequency of about 2 kHz to about8 kHz, or about 3 kHz to about 5 kHz, e.g., about 4 kHz. In someaspects, the method comprises using 90 degree pulses, e.g., ¹H 90 degreepulses about 2.5 μs. In some aspects, the ¹H CPMG spectra are obtainedwith about 5 to about 100 pi pulses (e.g., about 10 to about 90, about20 to about 80 pi pulses) of about 2 μs to about 20 μs (e.g., about 5μs, about 10 μs, about 15 μs, about 20 μs) in length. For example, eachof the pi pulses can be separated by 500 μs, and optionally, the totaltime of CPMG pulses is 10 ms. In exemplary aspects, the method comprisesobtaining a plurality of ¹H CPMG spectra while spinning at a frequencyof up to 5 kHz, above 8 kHz, or about 14 kHz. In some aspects, thecontact time of the ¹³C CP during the measuring is about 100 μs to about10 ms, e.g., about 250 μs, about 500 μs, about 750 μs, about 1 ms, about2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about 7 ms, about8 ms, about 9 ms, about 10 ms). In exemplary aspects, the measuringcomprised an RF spin lock pulse on ¹³C of about 20 kHz to about 100 kHz,e.g., about 50 kHz. In exemplary aspects, the ramp pulse on ¹H ismatched.

The method of detecting in exemplary embodiments in some aspectscomprises quantifying the content of crystalline biomolecules in thesample. In exemplary instances, the crystalline biomolecules exhibit aspectroscopic signature different than amorphous biomolecules. Inexemplary instances, the crystalline biomolecules exhibit aspectroscopic signature of higher molecular mobility than amorphousbiomolecules.

Accordingly, provided herein are methods of quantifying the content ofcrystalline biomolecules and/or amorphous biomolecules in a sample. Inexemplary aspects, the method comprises: (A) obtaining a plurality of ¹HNMR Carr-Purcell-Meiboom-Gill (CPMG) spectra of the sample, and (B)obtaining a ¹³C NMR cross-polarization (CP) spectra of the sample. Themethod of quantifying the content disclosed herein can be carried outaccording to the teachings in reference to methods of detectingcrystalline biomolecules and/or amorphous biomolecules in a sample.

In exemplary aspects of the methods of detecting or quantifying, thecrystalline biomolecules are bi-refringant and optionally, do notdiffract. In exemplary aspects, the crystalline biomolecules do notdiffract or poorly diffract at about 1 Å to about 2 Å. With regard tothe detecting methods described herein, the biomolecule can be anybiomolecule described herein, including, e.g., a polypeptide, protein,polysaccharide, lipid (e.g., glycolipid, phospholipid, sterol),polynucleotide or nucleic acid (e.g., DNA or RNA). In exemplaryembodiments, the biomolecule is a protein comprising one or morepolypeptide chains. In exemplary instances, the biomolecule is anantibody or immunoglobulin, or an antigen-binding antibody fragmentthereof, including any of those described herein under the sectionentitled “Biomolecules”, In exemplary instances, the protein comprisesone or more glycans, i.e., are glycanated biomolecules. In exemplaryembodiments, the biomolecules are glycanated antibodies.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1: Buffer Exchange of a Crystalline Monoclonal Antibody

The kSep® system (Sartorius Stedim North America, Inc., Bohemia, N.Y.)is a counterflow gradient centrifugation system designed for biologicsprocessing for harvesting cells as products or discarding cells andcollecting the supernatant as product during manufacturing. This systemis described in Kelly et al., Biotechnol. Prog. 32(6): 1520-1530 (2016)and U.S. Patent Publication No. 2011/0207222. The kSep® system differsfrom a traditional centrifugation system in that centrifugal forces arebalanced by opposing forces of a continuous flow of fluids (e.g., media,buffer), creating a fluidized bed system of cells or particles withinthe apparatus. See, Dechsiri, C. (2004). Particle transport in fluidizedbeds: experiments and stochastic models Groningen: s.n. In a traditionalcentrifugation unit, a packed-bed system is achieved. The kSep® systemalso utilizes single-use components minimizing the need for cleaning andis conducive to aseptic processing through sterile welding connections.The kSep® system is available as a laboratory- or production-scalesystem. The kSep®400 system is a laboratory scale system that has thecapacity to process 400 ml per cycle at a maximum flowrate of 114 L/hr.The kSep®400 system has four individual 100 ml chambers and each chambercan be run on its own or simultaneously with the others. The kSep®60005system is a production scale system (see FIG. 1) that has the capacityto process 6000 ml per cycle at a maximum flowrate of 720 L/hr. ThekSep®6000S system has six individual 1000 ml chambers and each chambercan be run on its own or simultaneously with the others.

While the kSep® system has been evaluated for its use in proteincrystallization (McPherson, Methods 34(3): 254-265 (2004), it has notbeen used for further downstream processing. Here, the kSep® system isused to replace a hypertonic crystallization buffer with an isotonicformulation buffer suitable for patient administration, following aprotein crystallization unit operation (see, e.g., FIG. 2). The kSep®system is also used in concentrating the protein crystallizationsuspension to a concentration amenable for patient dosing.

Using the kSep® system, a protein crystal suspension comprising acrystalline fully human monoclonal antibody was buffer exchanged withover 90% efficiency at one buffer exchange volumes and over 99%efficiency at two buffer exchange volumes. By altering the centrifugalforce and fluid flow force balance within the kSep® system, themonoclonal antibody protein crystal suspension was concentrated prior todispensing from the kSep® unit. Based on the amount of crystals loadedinto the unit and the fluid flow force relative to the centrifugalforce, the protein crystal suspension can be concentrated from 63 mg/mlto a range of 215 to 300 mg/ml. Counterflow gradient centrifugation viathe kSep® system is therefore an effective means to buffer exchangecrystallization buffer to formulation buffer and to concentrate proteincrystals to concentrations that are amendable for patientadministration.

Materials and Methods

Equipment: The kSep®400 unit was used for protein crystal suspensionbuffer exchange and concentration operations. The laboratory scale kSep®unit provided a platform to evaluate these operations without expendinglarge amounts of material. In order to reduce the usage of material forthe evaluation, the chamber usage was reduced from the standard fourkSep® system chambers which would use at a minimum 400 ml to either twoor one kSep® system chambers to reduce the volume usage to 100-300 ml.The concentrate-wash-harvest, CWH, disposable tubing set was used forthis evaluation as its configuration provided a proper avenue to harvestthe protein crystals after the buffer exchange and concentration unitoperations.

Materials: A fully humanized monoclonal antibody was used for thisevaluation. At the start of crystallization the antibody's formulationcontained 20 mM sodium acetate, 220 mM Proline, and 0.01% polysorbate 80at pH 5.0 with a concentration of 140 mg/ml. The crystallization bufferadded to the system contained 16 mM sodium phosphate, 20% PEG 3350 at pH8.4. The final protein crystal suspension at the conclusion of thecrystallization unit operation consisted of approximately 9 mM sodiumacetate, 98 mM Proline, 0.004% polysorbate 80, 7 mM sodium phosphate,8.9% PEG 3350 at pH 6.2. The crystal concentration was 62.2 mg/ml.

Buffer Exchange buffer: The buffer used for buffer exchange andconcentration was an isotonic buffer that consisted of the followingcomponents: 27 mM Succinic Acid, 15% PEG 3350, 0.1% PS80 at pH 5.2.

Results and Observations

Buffer Exchange

The buffer exchange operation was performed using the kSep® system'sconcentrate-wash-harvest pre-programmed application type (see, e.g.,FIG. 3). The critical parameters that impacted the overall bufferexchange were the wash flowrate and the number of volumes per chamber(equivalent to diavolumes). The wash flowrate parameter impacted bothprocessing time and buffer exchange efficiency. The higher the washflowrate, the shorter the processing time, but the lower the bufferexchange efficiency. In converse, a lower wash flowrate increased theprocessing time, but improved the buffer exchange efficiency. For thenumber of volumes per chamber, volumes was defined by the bioreactorvolume as specified in the kSep® system's recommended protocol. Toquantify buffer exchange efficiency, the pH of the outlet stream wasmeasured to observe the convergence of outlet pH with the bufferexchange buffer pH. The inlet feed material pH started at 6.2 and thebuffer exchange buffer pH was 5.2, therefore the buffer exchange wasconsidered complete once the outlet pH reached the pH of the bufferexchange buffer.

The results of the buffer exchange experiments are summarized inTable 1. Increasing the number of volumes per chambers improved thebuffer exchange efficiency as the outlet pH from experiment 1 was 5.34and the outlet pH from experiment 4 was 5.22. The other variables suchas wash flowrate and feed volume also impacted the final outlet pH, butnot to the same extent as the number of volumes per chamber.

TABLE 1 Buffer exchange experiments to evaluate key operating parametersFeed Wash 1 Inlet Buffer Volume Flowrate Centrifugation # (volumes)/Feed Outlet exchange Experiment (ml) (ml/min) Force (q) chamber pH pHbuffer pH 1 100 120 1000 1 6.2 5.34 5.2 2 100 70 1000 1 6.2 5.38 5.2 3300 120 1000 1 6.2 5.38 5.2 4 100 120 1000 2 6.2 5.22 5.2 5 200 120 10002 6.2 5.27 5.2

The kSep® system performed better in terms of buffer exchange efficiencywhen compared to a traditional bench top centrifuge as shown in FIG. 2.After a single spin and decant operation for the bench top centrifuge(equivalent to one number of volumes per chamber in the kSep® system),the outlet pH for a traditional centrifuge was 5.7 versus approximately5.3 for the kSep® system. Feed volumes of 100 to 300 ml, wash 1flowrates of 70 to 120 ml/min, and buffer exchange volumes of 1 to 2were evaluated and determined the extent to which complete bufferexchanged occurred.

Concentrate

Following the buffer exchange operation, the protein crystal suspensionwas concentrated by altering the balance between the centrifugationforce and the fluid flowrate. By having the centrifugation force greaterthan the fluid flowrate force, the protein crystals migrated towards oneend of the kSep® system chamber and effectively concentrated within thechamber. Methods to achieve this outcome included: increasing thecentrifugation force while keeping the fluid flowrate constant,decreasing the fluid flowrate while keeping the centrifugation forceconstant, or a combination of the previous two. There were two inputvariables that impacted the outlet concentration variable: washflowrate/centrifugation force and feed volume. Using the wash 2 flowratefunction with the kSep® system concentrate wash harvest program (see,e.g., FIG. 3), feed volumes from 150 to 300 ml and wash 2 flowrates of20 to 35 ml/min were evaluated. Based on those input variables,concentrations ranging from 168 to 303 mg/ml were achieved using thekSep® as shown in Table 2.

TABLE 2 Concentration experiments to evaluate key operating parametersCentrifu- Feed Wash 2 gation # Outlet Experi- Volume Flowrate Force(volumes)/ Concentration ment (ml) (ml/min) (g) chamber (mg/ml) 1 300 351000 1 303 2 200 35 1000 1 215 3 200 20 1000 1 235 4 200 35 1000 1 168 5150 20 1000 1 201

Previous experiments concentrated the protein crystal suspension tooutlet concentrations ranging from 122 to 229 mg/ml by altering the feedvolume from 260 to 500 ml and maintaining the wash 2 flowrate at 30ml/min. Even though outlet concentrations of greater than 200 mg/ml wereobserved, protein crystal agglomeration and crystallinity degradationwere also observed via microscope with birefringence capability due tothe use of a different Buffer Exchange buffer (10 mM NaPO4 pH6.2,10%PEG3350, 120 mM Lysine, 0.1%Ps80 buffer (Lysine)). The results fromthe initial failed experiments are shown in Table 3.

TABLE 3 Results from Initial Experiments Centrifu- Feed Wash 2 gation #Outlet Experi- Volume Flowrate Force (volumes)/ Concentration ment (ml)(ml/min) (g) chamber (mg/ml) 1 500 30 1000 0.6 215 2 400 30 1000 0.6 1783 120 30 1000 1.5 129 4 270 30 1000 0.6 122 5 300 N/A 1000 N/A 229 6 300N/A 1000 N/A 202

Harvest

After concentrating the protein crystal suspension to the desiredconcentration, the suspension must be dispensed (harvested) from thekSep® unit. Typically, in order to remove material from the kSep®system, the unit runs in reverse and pushes the material out through thekSep® chamber's inlet. The individual chamber peristaltic pumps pump inreverse to remove the material while the buffer pump pulses in buffer toprevent large pressure differentials from the inlet and outlet. Thepreprogrammed harvest operation in the kSep® concentrate-wash-harvestapplication is designed to harvest non-concentrated suspensions from theunit and as a result a number of initial experiments failed becauseeither material clogged the disposable tubing in the unit or the packedcrystal suspension was disrupted from the pulsing buffer pump or theindividual chamber peristaltic pump.

The initial experiments used the pre-programmed harvest operation with acentrifugation speed at 60 g and a harvest (i.e. chamber peristalticpump) flowrate at 50 ml/min. Using the pre-programmed operationsresulted a number of failures in which material could not be harvestedfrom the chamber. After a number of failed attempts at using thepre-programmed recipes, the harvesting operations was switched over tomanual control in order to manipulate the flowrates and valvepositioning on a real time basis. The centrifugation speed was decreasedand kept at a constant value in order to reduce the number of conditionsto evaluate and the buffer pump was kept on rather than pulsing buffer.The buffer pump was kept on so that there would be a constant increasein pressure from the flowrate of the buffer. The increase in pressurewould gradually force the packed crystal suspension out of the chamberwhile the individual chamber peristaltic pumps would slowly pullmaterial out of the crystal and prevent cavitation of the packed crystalsuspension. Disrupting the packed crystal suspension would negativelyimpact the final crystal concentration. Table 4 summarizes experimentsrelating to harvesting packed (concentrated) crystal suspension from thekSep® system. Table 4 documents some of the harvest experiments and theobservations noted during the experiment. The packed crystal suspensioncould not be harvested with the preprogrammed harvest automation as itwas currently setup. The harvest operation needed to be switched into amanual operation in which the experimenter can adjust settings on a realtime basis rather rely on automated controls. Experiments that aredesignated with an “a” and “b” are part of the same experiment whereconditions were adjusted following a failed harvest attempt.

TABLE 4 Chamber Pump Buffer Pump No. of Centrifugation Flowrate FlowrateChambers Expt Program Speed (g) (ml/min) (ml/min) in Use Observation 1 Harvest 60 50 N/A—controlled 1 Material clogged Automation by automationin chamber 2a Harvest 80 50 N/A—controlled 1 Material clogged Automationby automation in chamber 2b Manual 8 75 25 1 Material removedIntervention from kSep chamber 3a Manual 8 75 25 1 Material cloggedIntervention in chamber 3b Manual 8 75 50 1 Material removedIntervention from kSep chamber 4  Manual 8 75 50 2 Uneven harvestIntervention of chambers 5  Manual 8 75 25 3 Disruption of packedIntervention crystal suspension during harvest 6  Manual 8 75 25 1Material removed Intervention from kSep chamber 7  Manual 8 75 25 1Material removed Intervention from kSep chamber 8  Manual 8 75 25 1Material removed Intervention from kSep chamber

Another factor to consider is the individual chamber pumps; theindividual chamber pumps need to pull the material out of the chamberwithout causing any cavitation within the packed crystal suspension.Cavitation disrupts the concentration profile and creates a non-uniformconcentration gradient across the concentrated crystal suspension asdocumented in Table 4 experiments 4 and 5. To harvest the concentratedcrystal suspension, one chamber was harvested at a time. Harvestingmultiple chambers at once increased the likelihood of cavitation. Thecentrifugation force was kept at 8 g, the buffer pump was set from 25-50ml/min and the individual chamber pump was set to 75 ml/min (FIG. 4).The centrifugation force and buffer pump create the driving force topush the material out of the chamber while the individual chamber pumpacts as a pulling mechanism to ensure cavitation is limited.

Example 2: Distinguishing

Washing of samples in Formulation Buffer: Samples of crystallinematerial were prepared in a batch method as essentially described inInternational Patent Application Publication No. WO2016010927, entitled“CRYSTALLINE ANTIBODY FORMULATIONS” which is incorporated by referencein its entirety.

All samples were washed in a formulation buffer of 27 mM succinic acidand 15% PEG 3350 and 0.1% Tw-80, pH=5.5. One mL of the crystals oramorphous material was combined with 1 mL of formulation buffer in anEppendorf tube and mixed by gentle inversion. The mixture was thencentrifuged for 5 mins at 2000 RPM=376 rcf and the supernatant removed.Another 1 mL of formulation buffer was added and the pellet resuspendedand mixed gently followed by centrifugation. This was repeated once.

Sample Packing: All samples were packed in 4.0 mm Bruker rotors. A 1 mLpipet tip was cut to act as a funnel into the rotor and sized so thatthe rotor and funnel fit inside a 2.0 mL Eppendorf tube. Samples werepacked by adding 100 μL of the suspension to the funnel and thencentrifuged at 10 k rcf for 2 mins. This was repeated 4-8 times untilthe rotor was well-packed with solid sample.

SSNMR: A Bruker Ascend Avance III widebore spectrometer operating at ¹Hresonance frequency of 500 MHz was used for analysis. A 4 mm H/F/X MASprobe operating at a spinning frequency of 4 kHz was used for allexperiments, except when noted otherwise. A BCU II −80/60 temperatureunit was used to control temperature to 300 K, except when doingtemperature studies. ¹H 2.5 us pi/2=160 W were used for 90 degreepulses. ¹H CPMG spectra were collected with 20 pi pulses of 5 μsseparated by 500 μs for a total 10 ms of CPMG pulses suppressing signalsfrom solid material. After measuring the CPMG spectra the samples werespun up to 14 kHz and a 13C cross polarization (CP) was measured as away to calibrate the total solid material in the rotor and account fordifferences in packing between samples and rotors. The CP spectra used a2 ms contact time and 50 kHz RF spin lock pulse on 13C and appropriatelymatched ramp pulse on ¹H.

Observations

The CPMG spectra were used to suppress proton signals arising from partsof the sample that are in the solid state and not mobile. In the CPMGspectra of the mAb crystals and amorphous product, there were somesignals that appeared in the crystalline material that did not appear inthe amorphous material (see FIG. 5). These signals could be used toquantify the amount of crystalline content as shown with the spectrafrom samples spiked with either 5% crystalline or 5% amorphous mAbs(FIG. 6). The signals in the crystalline spectra also increased withincreasing temperature, indicating that those parts of the molecule werebecoming more mobile (FIG. 7).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the disclosure (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope of the disclosureunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the disclosure.

Preferred embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure.Variations of those preferred embodiments can become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to use such variations as appropriate,and the inventors intend for the disclosure to be practiced otherwisethan as specifically described herein. Accordingly, this disclosureincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the disclosure unless otherwiseindicated herein or otherwise clearly contradicted by context.

1. A method of preparing a composition comprising crystallinebiomolecules, comprising: a) forming a fluidized bed of crystallinebiomolecules in a rotating chamber comprising an inlet and an outlet,wherein the fluidized bed is created by rotating the chamber about asubstantially horizontal axis to create a centrifugal force(F_(centrifugal)) in said chamber, flowing a first stream of a firstsolution through the inlet in a direction opposite to the direction ofthe F_(centrifugal) and at a first flow rate (FR1) having a force(F_(FR1)) which counter balances F_(centrifugal), and collecting thefirst solution from the chamber while substantially maintaining theformation of the fluidized bed of crystallized biomolecules; b)performing step (i), (ii), or (iii): i. flowing a second stream of asecond solution to replace the first stream of the first solution whilesubstantially maintaining the formation of the fluidized bed ofcrystallized biomolecules; ii. concentrating the crystallizedbiomolecules within a region of the chamber by changing FR1 to a secondflow rate (FR2) having a force (F_(FR2)) which is less thanF_(centrifugal) or by increasing the speed of rotation of the chamber toincrease F_(centrifugal) to a level which is greater than F_(FR1); iii.a combination of step (i) and step (ii); and c) flowing a second streaminto the chamber through the outlet in a direction which is parallel tothe direction of the F_(centrifugal) to remove the crystallinebiomolecules from the chamber.
 2. The method of claim 1, whereinF_(centrifugal) is 1000 g during at least step (i).
 3. The method ofclaim 1, wherein the feed volume of the second stream of the secondsolution is about 50 to about 500 ml during step (i).
 4. (canceled) 5.The method of claim 1, wherein (A) the number of volumes of the firstsolution per chamber is 2 or more during step (i), (B) FR1 is about 50ml/min to about 150 ml/min during step (i), (C) the first solution is ahypertonic crystallization buffer, (D) the second solution is anisotonic formulation buffer, (E) the second solution is an isotonicformulation buffer, (F) the F_(centrifugal) is 1000 g during step (ii),(F) the feed volume of the second stream of the second solution is about100 to about 500 ml during step (ii), (G) the number of volumes of thefirst solution per chamber is 1 or 2 during step (ii), (H) FR2 is about10 ml/min to about 50 ml/min during step (ii), (I) the concentration ofthe crystalline biomolecules is increased at least 2-fold, (J) themethod comprises decreasing F_(centrifugal) to a level of about 5 g andabout 20 g, (K) FR1 is controlled by a first pump and the first pump isset to about 10 ml/min to about 100 ml/min after step (b), (L) therotating chamber is connected to a chamber peristaltic pump. (M) themethod is carried out in an apparatus comprising more than one rotatingchamber, (N) the crystalline biomolecule is a protein or comprises oneor more polypeptide chains, or (O) any combination thereof. 6.-16.(canceled)
 17. The method of claim 5, wherein the concentration of thecrystalline biomolecules is increased at least 3-fold or at least4-fold. 18.-23. (canceled)
 24. The method of claim 5, wherein thechamber peristaltic pump is set to pump in the same direction of thefirst pump and F_(centrifugal).
 25. (canceled)
 26. (canceled)
 27. Themethod of claim 5, wherein the chamber peristaltic pump is set to about75 ml/min, the first pump is set to about 50 ml/min, and F_(centrifugal)is decreased to below 10 g.
 28. (canceled)
 29. The method of claim 5,wherein the apparatus comprises at least 2, at least 4 or at least 6rotating chambers.
 30. (canceled)
 31. The method of claim 29, whereinsteps (a) to (c) are carried out in more than one chamber.
 32. Themethod of claim 31, wherein crystalline biomolecules are removed from achamber of the apparatus at a time distinct from when crystallinebiomolecules are removed from another chamber of the apparatus. 33.(canceled)
 34. (canceled)
 35. A method of detecting crystallinebiomolecules and/or amorphous biomolecules in a sample, the methodcomprising: a) obtaining a plurality of ¹H NMR Carr-Purcell-Meiboom-Gill(CPMG) spectra of the sample, and b) obtaining a ¹³C NMRcross-polarization (CP) spectra of the sample.
 36. The method of claim35, comprising (A) operating a ¹H resonance frequency of about 250 toabout 1000 MHz during step (a) of the method, (B) maintaining thetemperature at about 250 to about 350 K, (C) operating a magic anglespinning (MAS) probe, or (D) any combination thereof.
 37. (canceled) 38.(canceled)
 39. The method of claim 36, wherein the MAS probe (A)comprises at least 2 RF channels, (B) is tuned to ¹H and ¹³C, (C)operated with a spinning frequency of about 2 kHz to about 8 kHz. 40.(canceled)
 41. (canceled)
 42. The method of claim 35, comprising using90 degree pulses, optionally, ¹H 90 degree pulses of about 2.5 μs. 43.(canceled)
 44. The method of claim 35, wherein the ¹H CPMG spectra wereobtained with about 5 to about 100 pi pulses of about 2 μs to about 20μs in length, optionally, wherein each of the 20 pi pulses wereseparated by about 10 μs to about 1 ms.
 45. (canceled)
 46. (canceled)47. The method of claim 35, comprising obtaining a plurality of ¹H CPMGspectra while spinning at a frequency of up to 14 kHz.
 48. The method ofclaim 35, wherein the contact time of the ¹³C CP during the measuring isabout 100 μs to about 10 ms.
 49. (canceled)
 50. (canceled)
 51. Themethod of claim 35, comprising quantifying the content of biomoleculesin the sample.
 52. The method of claim 35, wherein the crystallinebiomolecules exhibit a spectroscopic signature different than amorphousbiomolecules or the crystalline biomolecules exhibit a spectroscopicsignature of higher molecular mobility than amorphous biomolecules. 53.(canceled)
 54. The method of claim 35, wherein the crystallinebiomolecules are bi-refringant, do not diffract, comprise proteins orone or more polypeptide chains. 55.-58. (canceled)